1. Field of the Invention
The present invention relates to neutralizing antibodies targeting high mobility group box 1 (HMGB1), and compositions and methods comprising such antibodies useful in treating, inhibiting or preventing HMGB1-associated neuropathy. The invention also relates to the combination of the variable regions of heavy and light chain immunoglobulins derived from a single human antibody-producing cell and amino acid molecules encoding such antibodies and antigen-binding portions, methods of making human anti-HMGB1 antibodies and antigen-binding portions, compositions comprising these antibodies and antigen-binding portions and methods of using the antibodies, antigen-binding portions, and compositions.
2. Description of Related Art
In normal physiological conditions, HMGB1 is confined to the nucleus as a non-histone DNA binding protein that is involved in regulation of chromatin dynamics (Hock et al., 2007), such as stabilizing nucleosome formation (Goodwin et al., 1973), facilitating gene transcription (Kleinschmidt et al., 1983) and modulating steroid hormone receptors (Boonyaratanakornkit et al., 1998). In contrast, in the case of tissue injury, HMGB1 migrates toward the cytoplasm and then is released extracellularly, acting as a danger signal (Bianchi and Manfredi, 2007). In addition to being passively released by necrotic cells, HMGB1 can also be actively secreted by specific cells, e.g., macrophages and myeloid cells, of the innate immune system activated by pro-inflammatory signals such as lipopolysaccharide, tumor necrosis factor-α (TNF-α) and interleukin-1 β(IL1-β) (Bonaldi et al., 2003). It has been also demonstrated as a cytokine that mediates endotoxin lethality (Wang et al., 1999), propagating inflammation (Wang et al., 2001), prolonging macrophage activation (Andersson et al., 2000) and chemoattracting mesoangioblast (Palumbo et al., 2004). It is becoming increasingly clear that both passively released and actively secreted extracellular HMGB1 convey the same danger signal to attract and activate inflammatory cells (Abraham et al., 2000; Andersson et al., 2000), to enhance the expression of cell adhesion molecules in endothelial cells (Fiuza et al., 2003) and to impair the barrier function of endothelium (Yang et al., 2006). Specifically, serum levels of HMGB1 have been directly associated with mortality in patients with lethal sepsis and in experimental models of endotoxaemia (Wang et al., 1999; Fink, 2007), suggesting that HMGB1 is a crucial late mediator of the sustained activation of pro-inflammatory cascades associated with fatal outcomes. Specific receptors interacting with extracellular HMGB1 include receptor for advanced glycation end products (RAGE), toll-like receptor (TLR)-2 and TLR-4 (Hori et al., 1995; Park et al., 2004). The binding of HMGB1 to its receptors results in the activation of several kinases such as extracellular signal-regulated kinases (ERK)-1/2, p38 mitogen-activated protein (MAP) kinases, and c-Jun NH2-terminal kinase, which ultimately leads to the activation of nuclear factor-κB (NF-κB)-dependent genes (Huttunen et al., 1999; Fiuza et al., 2003).
As human HMGB1 has cytokine activities and mediates prolonged systematic inflammation as well as immune responses, we have thus investigated and successfully established the link between an aberrant production of HMGB1 in plasma of patients with neuromyelitis, i.e., neuromyelitis optica (NMO) and conventional multiple sclerosis (MS) (Wang et al., 2012). NMO and MS are a chronic, inflammatory, demyelinating disease of the central nervous system (CNS), most frequently starting with a series of bouts, each followed by complete remission and then a secondary, progressive phase during which the neurological deficit increases steadily. However, the clinical course of NMO is usually more severe than classical or conventional MS (Cornelio et al., 2009). Within five years of onset, fifty percent of NMO patients either loss of functional vision in at least one eye or becoming unable to walk unassisted, or the annual costs for hospitalization exceed several hundred million. There remains an urgent need for prophylactic, pre-emptive and treatment strategies for management of HMGB1-associated neuromyelitis.
Anti-huHMGB1 therapies by using of monoclonal antibodies (mAbs) to specifically bind to and neutralize this particular target protein are expected to be effective in nature. Accordingly, targets with highly selective late mediator are preferred to broaden the therapeutic window (Wang et al., 1999). Furthermore, given the remarkable divergent emergence of the physiological from pathological conditions of huHMGB1, mAbs raised against it are expected to selectively target extracellular HMGB1. Based on these considerations, a monoclonal antibody against huHMGB1 that may inhibit receptor interaction and therefore block kinase activation and the downstream pro-inflammatory response, e.g., TNF-α secretion is highly desirable.
Although mAbs are long-established as essential research tools, their therapeutic promise has taken considerably longer to realize, requiring further advances, such as the humanization of mouse antibodies and/or transgenic protocols, to reduce their immunogenicity inherent in murine proteins. The biopharmaceutical industry has thus seen a full shift of new antibody therapeutics from chimeric to humanized to fully-human sequences. The ultimate objective in the industry is to manufacture a mAb drug that is identical to that which is produced in the human body.
In theory, heavy and light chain components derived entirely from the human origin could be used to assemble mAbs. Indeed, the use of phage antibody technology to construct human heavy and light chain libraries as described in e.g., WO92/01047, U.S. Pat. Nos. 5,652,138 and 5,885,793A, offers the ability to isolate such human antibodies directly. Moreover, U.S. Pat. Nos. 5,652,138 and 6,075,181 demonstrate the feasibility to construct in vivo transgenes to introduce into a non-human animal substantially the entire human immunoglobulin loci and subsequently perform animal immunization to obtain heavy-and-light-chain pairs comprised human sequences. However, the resultant pairings may not be naturally-occurring and still remain to be immunogenic as indicated in the official FDA labeling information (http://www.accessdata.fda.gov)—the frequencies of patient to generate anti-drug antibodies can be as high as 26% and 3.8% for Humira (adalimumab) and Vectibix (panitumumab), respectively. Therefore, this may not be the best available strategy. In fact, while there are general rules (De Groot and Scott, 2007), predicting the precise immunogenicity of a specific protein in the genetically heterogeneous human population is a difficult if not a fundamentally impossible task. This is highlighted again by the case of Enbrel (etanercept), which is a biopharmaceutical produced by recombinant fusion between the TNF receptor and the constant region of the IgG1 antibody to act as a TNF inhibitor. Since both fusion partners are bona fide naturally-occurring ubiquitous proteins in humans and, in principle, should not be immunogenic. Yet clinical data specify that 5.6% treated patients tested positive for anti-etanercept antibodies (Dore et al., 2007).
Our understanding in all of the area of immunogenicity is increasing, but currently it seems impossible to close the circle. Alternatively, a sharp clinical contrast provided by the therapeutic immunoglobulin preparations, and especially intravenous immunoglobulin (IVIg), may well turn out to be optimistic in resolving the problem of immunogenicity. The IVIg refers to a therapeutic biological product containing human IgG that is prepared by large-scale industrial fractionation of human plasma derive from samples collected from thousands of blood donors (Kazatchkine and Kaveri, 2001; Seite et al., 2008), and thus represents in essence a preparation of human polyclonal antibodies. It usually administrated 400 milligrams to 1 gram per kilogram body weight weekly, by which it generally acts as a replacement therapy for many years (Farrugia and Poulis, 2001; Wittstock et al., 2003). Despite the worldwide consumption of IVIg may well above 100 tonnes per year, adverse reactions to IVIg occur in less than five percent of patients (http://consensus.nih.gov/1990/1990IntravenousImmunoglobulin080html.htm), also patients seem to tolerate to IVIg and very few if there is any anti-IVIg responses reported (Lemieux et al., 2005; Imbach, 2012b; Imbach, 2012a). Lack of immunity to IVIg suggests that ingenious designs to harness naturally-occurring configurations inherent in a single antibody-producing B cell could provide a substitute for such approach.
Nevertheless, while foreign antigens, which are molecules derived from a potentially harmful invader, trigger the production of antibodies by the immune system; self antigens such as HMGB1 are usually tolerated by the immune system. In reality, although self antigen-reactive B cells may be present in the body, self antigens are not likely to initiate an immune response and thus without leading to the production of specific antibodies. To effectively transform such challenging circumstances, “site-directed in vitro immunization” (Chin et al., 2007)(U.S. Pat. Nos. 7,494,779, 8,021,860 and 8,158,386) was developed by the inventors to initiate in vitro self antigen-specific immune responses from primary human lymphoid cells. Self antigens can then be neutralized by the resultant mAbs similar to foreign ones.
One of the major problems in obtaining highly potent human therapeutic mAbs is how to isolate antigen-specific clones derived from the site-directed in vitro immunization scheme or even the circulating memory B cell compartment of a human donor. This so-called rare population are clones that under-representative and, accordingly, very little in number. Some of these rare specific cells are expected to be most interesting in modern medicine and thus much research has attempted to address this question, including the use of the antigen-conjugated chitosan as taught by WO 2010104828. However, in light of the limitations of pH-dependent solubility and the reactivity of its amine groups in enzymatic conjugation inherited in biopolymer chitosan to account for many aspects of protein conjugation, it will clearly be important to develop a method for the separation of immune cells by interactions of proteins of said B cells with antigens.
Rabbit antisera have been generated against a synthetic peptide corresponding to the amino terminal part of HMGB1 (amino acid residues 2-15), coupled to radially branching lysine dendrites and against the entire HMGB1 (U.S. Pat. No. 6,468,533). Murine mAbs produced from a hybridoma resulting from the fusion of mouse myeloma with B cells obtained from a mouse immunized with a part of huHMGB1 (amino acid residues 89-1162) or purified recombinant rat HMGB1 (U.S. Pat. No. 7,288,250). Rat mAbs specific to the carboxyl terminal (amino acid residues 200-211) were reported using HMGB1/HMGB2 as an immunogen (Liu et al., 2007). R&D Systems, Inc. makes and sells a polyclonal anti-human HMGB1 antibody (Cat. # AF 1690) produced in chicken immunized with purified, NS0-derived recombinant human HMGB1.
To date, no complete human mAbs with naturally-occurring configurations inherent in a single antibody-producing B cell to HMGB1 neither have the use of such kind of mAbs for inhibiting or treating HMGB1-associated neuromyelitis been reported.